I. Sequence Analysis Using Dideoxynucleotides
Chain terminating 2',3'-dideoxynucleotide triphosphates (ddNTPs) are widely used in nucleic acid sequencing technology (Sanger, et al., Proc. Nat. Acad. Sci.(USA), 74:5463 (1977)). Traditionally, Sanger sequencing involves the polymerase-mediated incorporation of a dideoxynucleotide onto the 3' end of an elongating DNA chain. Incorporation of a dideoxynucleotide results in chain termination, as dideoxynucleotides lack a 3'-OH suitable for further elongation. Methods for Sanger sequencing are disclosed, for example, in J. Sambrook, et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), and in J. W. Zyskind, et al., Recombinant DNA Laboratory Manual, Academic Press, Inc., New York (1988)), both herein incorporated by reference.
In Sanger sequencing, four reactions are typically performed for each polynucleotide to be sequenced. Each reaction contains a DNA template, an oligonucleotide primer, and a mixture of all four deoxynucleotide triphosphates (dNTPs). In addition, each reaction contains one of the four ddNTPs. Either the oligonucleotide primer or the ddNTPs are radiolabeled. The reaction results in a series of radioactivly labeled, prematurely terminated extension reaction products, which are then separated on individual lanes of a high resolution polyacrylamide gel. The gel is then dried and exposed to photographic or x-ray film to record the results for analysis. The procedure is time-consuming, requires significant technical expertise, and involves the use of both radioactive and neurotoxic materials.
The original chain termination or Sanger method has been improved in several ways, and serves as the basis for all currently available automated DNA sequencing machines. See, e.g., Sanger et al., J. Mol. Biol. 143:161-78 (1980); Schreier et al., J. Mol. Biol. 129:169-72 (1979); Smith et al., Nucleic Acids Res. 13:2399-2412 (1985); Smith et al., Nature 321:674-79 (1987); Smith et al., U.S. Pat. No. 5,171,534; Prober et al., Science 238:336-41 (1987); Section II, Meth. Enzymol. 155:51-334 (1987); Church et al., Science 240:185-88 (1988); Swerdlow et al., Nucleic Acids Res. 18:1415-19 (1989); Ruiz-Martinez et al., Anal. Chem. 65:2851-58 (1993); Studier, Proc. Natl. Acad. Sci. (USA) 86:6917-21 (1989); Kieleczawa et al., Science 258:1787-91; and Connell et al., BioTechnigues 5:342-348 (1987)).